Successful cryopreservation of rat pronuclear-stage embryos by rapid cooling

Embryo cryopreservation is a valuable tool for efficient production of animals as well as banking of genetic resources. Even though the laboratory rat is one of the most important experimental animals for various research fields, it has been reported that survival and developmental ability of cryopreserved rat embryos are generally low, especially at the early stages. The aim of the present study was to establish rapid cooling method that can be applied for cryopreservation of rat pronuclear-stage embryos using Cryotops (a device). First, optimal equilibration time was examined. Pronuclear-stage embryos were equilibrated in 7.5% ethylene glycol (EG)+7.5% dimethylsulfoxide (DMSO)+20% fetal calf serum (FCS) for 7, 8 or 9 min at 20-22 degrees C and then 15% EG+15% DMSO+0.5M sucrose+20% FCS for 1 min at 20-22 degrees C, being plunged into liquid nitrogen on Cryotops. This established that development to the 2-cell (82.0+/-9.7% to 96.1+/-3.0%) and blastocyst (36.5+/-2.1% to 40.3+/-10.2%) stages in vitro was not influenced by the equilibration time. Furthermore development to term in vivo (56.0+/-4.9%) was equivalent to the rate (54.8+/-6.6%) obtained with control embryos. Taken together, this demonstrated that this method is suitable for the successful cryopreservation of pronuclear-stage embryos in rats.